Picornaviruses include inter alia polioviruses, which cause infantile paralysis, and rhinoviruses, which cause the common cold. Picorna-related viruses, which replicate by mechanisms similar to picornaviruses, include hepatitis A and C, major causes of human hepatitis. Although poliovirus vaccines are available, cases of polio still develop where vaccination is not properly used. Vaccines for other picornaviruses may not be feasible, for instance, due to the high rate of mutability of the viral coat proteins in the rhinoviruses. Therefore, there is a need for methods and compositions for selectively inhibiting picornavirus replication without toxic effects on the host cells.
Poliovirus, the prototype member of the picornaviridae family, is a single stranded, plus-sense RNA virus which multiplies in the cytoplasm of infected cells. The RNA genome comprises approximately 7,500 nucleotides and codes for a 250 kDa polyprotein (Kitamura, N. et al. Nature (1981) 291:547-553 and Racaniello, V. R., et al. Proc Natl Acad Sci USA (1981) 78:4887-4891. The unusually long 5' untranslated region (5'UTR) of poliovirus RNA (750 nucleotides) is highly structured (Skinner, M. A. et al. J Mol Biol (1989) 207:379-392; Agol, V. Adv Virus Res (1991) 40:103-180) and contains six to eight upstream AUGs, none of which appears to be used in initiation of translation (Pelletier, J. et al. J Virol (1988a) 62:4486-4492.
Translation of most mammalian cellular mRNAs proceeds by binding of ribosomes to the 5' cap structure followed by scanning of the mRNA until the appropriate AUG is encountered by the ribosome (Kozak, M. Microbiol Rev (1983) 47:1-45). In contrast translation of naturally uncapped poliovirus RNA has been shown to be mediated by a mechanism involving internal entry of ribosomes near the initiator AUG (Pelletier, J. et al. Nature (1988) 334:320-325). Recent studies have demonstrated that internal entry of ribosomes requires an element located between nucleotides 320-631 within the 5'UTR of poliovirus RNA (Pelletier, J. et al., supra). This sequence element has been termed a ribosome landing pad (RLP) or, more generally, internal ribosome entry site (IRES). Although a number of cellular polypeptides have been implicated in IRES-dependent translation, the precise mechanism of internal initiation of translation remains poorly understood.
In addition to poliovirus many other picornaviruses have been shown to utilize this novel mechanism for initiation of translation (Jang, S. K. et al. Genes Dev (1990) 4:1560-1572, Belsham, G. J. et al. J Virol (1990) 64:5389-5395, Jackson, R. et al. Trends Biochem Sci (1990) 15:477-483, Luz, N. et al. FEBS Letters (1990) 269:311-314, Luz, N. et al. Virology (1991) 65:6486-6494, Bandopadhyay, P. K. et al. J Virol (1992) 66:6249-6256, Borman, A. et al. Virology (1992) 188:685-696, Borman, A. et al. Gen Virol (1993) 74:1775-1788). The RNA genomes of two picorna-related viruses, hepatitis A and C, have been shown to utilize internal ribosome entry for translation initiation (Kohara, K. T. et al. J Virol (1992) 66:1476-1483 and Glass, M. J. et al. Virology (1993) 193:842-852). Two cellular mRNAs, encoding immunoglobulin heavy chain binding protein (Bip), the mouse androgen receptor (32) and the antennapedia of Drosophila, also have been shown to use internal initiation of translation (Macejak, D. G. et al. Nature (1991) 353:90-94 and Oh, S. K. et al. Genes Dev (1992) 6:1643-1653).
All picornaviral mRNAs that utilize IRES-dependent translation contain a polypyrimidine tract located at the 3'-border of the IRES sequences within the 5'UTR. Recent evidence indicates that proper spacing between the polypyrimidine tract and the cryptic AUG at nucleotide 586 of the poliovirus 5'UTR is important for viral translation (Jackson et al. (1990, supra), Jang et al. (1990, supra), Pilipenko, E. V. et al. Cell (1992) 68:119-131).
Accurate translation of poliovirus mRNA in rabbit reticulocyte lysate requires HeLa cell proteins, indicating involvement of cellular proteins in internal initiation of translation (Brown, B. A. et al. Virology (1979) 97:376-405; Dorner, H. A. et al. J Virol (1984) 50:507-514) . A 50 kDa protein has been shown to interact with the RNA stem-loop structure located between nucleotides 186-221 in poliovirus type 1 RNA (Najita, L. Proc Natl Acad Sci USA (1990) 87:5846-5850). The physiological significance of this binding is yet unclear.
Another protein called p52, more abundant in HeLa cells than in rabbit reticulocytes, has been found to specifically bind to the stem-loop structure between nucleotides 559-624 of type 2 poliovirus RNA (Meerovitch, K. et al. Genes Dev (1989) 3:1026-1034). This p52 protein appears to be identical to the human La auto antigen (Meerovitch, K. et al. J Virol (1993) 67:3798-3807). This nuclear protein, which is recognized by antibodies from patients with the autoimmune disorder lupus erythematosus, leaches out of the nucleus into the cytoplasm in poliovirus-infected HeLa cells. Cell extracts immunodepleted with La antibodies fail to promote cap-independent translation and exogenous addition of purified La protein corrects aberrant translation of poliovirus RNA in reticulocyte lysate which contains little or no p52 (Meerovitch et al. (1993, supra).
UV crosslinking studies have demonstrated another cellular protein, p57 to interact with IRES elements of encephalomyocarditis (EMC), foot-and-mouth disease, rhino-, polio- and hepatitis A viruses (Jang et al. 1990, supra; Borovjagin, A. V. et al. Nucleic Acids Res (1991) 19:4999-5005; Luz et al. 1991, supra; Pestova, T. V. et al. J Virol (1991) 65:6194-6204; Borman et al. 1993, supra, and Chang, K. H. et al. J Virol (1993) 67:6716-6725). It has been demonstrated recently that p57 binding to an IRES of EMCV is identical to that of a polypyrimidine tract binding protein (PTB), which presumably plays a role in a nuclear splicing (Hellen, C. U. T. et al. Proc natl Acad Sci USA (1993) 90:7642-7646). Anti-PTB antibody inhibits translation of EMCV and poliovirus RNA and, therefore, PTB may be directly involved in IRES-directed translation.
In addition two other cellular proteins with molecular weights of 38 and 48 kDa have been shown to specifically interact with RNA structures spanning nucleotides 286-456 of poliovirus. These two proteins are reported to be present in HeLa cells in higher quantities than in reticulocyte lysate and appear to be involved specifically in poliovirus translation (Gebhard, J. R. et al. J Virol (1992) 66:3101-3109). Another 54 kDa protein cross-links to a region between nucleotides 456-626 and is required for translation of all mRNAs (Gebhard et al. 1992, supra). A recent report indicates the role of a 97 kDa protein in IRES-dependent translation of human rhinovirus RNA (Borman et al. 1993, supra). RNA-protein complex formation has also been demonstrated with the regions encompassing nucleotides 98-182 and 510-629 of the poliovirus RNA (del Angel, P. A. G. et al. Proc Natl Acad Sci USA (1989) 86:8299-8303).
Taken together, the results above are compatible with a mechanism of picornaviral translation that involves direct interaction between cellular factors and RNA sequences and/or secondary structures leading to internal initiation. The action of the binding proteins in this mechanism is not known, but transacting proteins may direct ribosomes to enter the mRNA or may alter RNA structure to facilitate ribosome binding.
In a previous study the present inventors have shown that yeast cells are incapable of translating poliovirus RNA both in vivo and in vitro and that this lack of translation represents selective translation inhibition which requires the 5'UTR of the viral RNA (Coward, P. et al. J Virol (1992) 66:286-295). The inhibitory effect was found to be due to a transacting factor present in yeast lysate that can also inhibit the ability of HeLa cell extracts to translate poliovirus RNA. Initial characterization of this inhibitor showed that its activity was heat stable, resistant to proteinase K digestion, phenol extraction and DNase digestion, but sensitive to RNase (Coward et al. 1992, supra).